The present invention relates to a rapid, high performance assay for the detection of pathogenic E. coli which is based on TaqMan(trademark) PCR technique, and to specific optimised oligonucleotide primers and labelled oligonucleotide probes useful in the assay.
Enterohemorrhagic, shiga-like toxin (sit) producing Escherchia coli (EHEC) have recently been recognized as an important human and animal pathogen (1-7). EHEC has been responsible for several food-borne outbreaks (8). The most notable were a multistate outbreak associated with a fast food chain in the western states of the USA with more than 600 individuals affected and 3 deaths in Washington (9), and an epedemic occurence in Japan with more than 6000 patients and approx. 8 fatal cases (10). Infection with EHEC causes diarrhea, hemorrhagic colitis, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome (HUS) that is characterised by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. HUS ultimately can result in a fatal outcome in affected children and immunocompromised individuals (3,11-17). Recently, in the South-Eastern parts of Germany (Bavaria) an increase of EHEC cases was reported during October 1995 and July 1996 with at least 45 severe infections leading to HUS accompanied by 7 deaths (18). Estimating that approx. 1 out of 15 EHEC infections results in HUS approx. 600-700 affected individuals might be assumed.
In most outbreaks reported, consumption of contaminated ground beef has been the source of infection (5,8,19-22), whereas in Japan raddish sprouts are suspected (10). EHEC has been isolated from cow milk (6,19,23), water (19), chicken, pork, and apple cider (19,24,25), but also human horizontal smear infections have been reported (15). Cattle appear likely to be the reservoir (22,26). Cross contamination, improper handling, and inadequate cooking all contribute to food-borne infections caused by EHEC. EHEC produce Shiga-like toxins (slt), also known as verotoxins or cytotoxins (12,27). A large proportion of EHEC have been found to belong to the serogroup O157:H7, but notably, also a variety of EHEC belonging to other serogroups (O22, O26, O55, O111, O114, O145) have been reported especially in Europe (12,15,28-32).
Besides EHEC, certain other strains of E. coli can cause enteritis or gastroenteritis and are grouped in enterotoxigenic strains (ETEC) (33-36), enteropathogenic strains (EPEC) (37), enteroinvasive strains (EIEC) (38,39), and enteroaggregative strains (EaggEC) (40,41). These strains are important pathogens and also pose severe public health problems. The diagnosis of these pathogens is vastly neglegted due to the lack of specific and sensitive routine test methods. ETEC synthesize heat labile and/or heat stable enterotoxins that can cause a secretory diarrhea (xe2x80x9ctraveller""s diarrheaxe2x80x9d) resembling that of Vibrio cholerae (36,42,43). Surface attachment of the ETEC organisms to the intestinal epithelial cell is a prerequisite to toxin production. Toxin production is plasmid mediated and most commonly involves E. coli serogroups O6, O15, O124, O136, O143, O145, and 0147 (32). EPEC cause diarrheal symptoms primarily in infants (32). Although the pathogenesis is unclear, the epithelial degradation of the gut, and the inflammatory response that are observed in tissue sections may be a consequence due to the adhesive properties of the bacterium. Specific attachment factors of EPEC are plasmid encoded (EAF=EPEC adherence factor) (37,44). EHEC often contain an adherence factor closely related to EAF that is known as ene (EHEC attaching and effacing gene) (45,46). EPEC most often belong to serogroups O6, O8, O25, O111, O119, and O142 (32).
EIEC strains are capable of penetrating and invading the intestinal epithelial cells and produce an inflammatory diarrhea similar to that caused by Shigella bacteria (38,47,48). Fecal smears contain blood, mucus and segmented neutrophils. EIEC contain virulence plasmids coding for additional pathogenic factors (48). Serogroups O28, O112, O115, O124, O136, O143, O145, and O147 are most commonly found on EIEC (32).
EaggEC are associated with persistent diarrhea in children and with traveller""s diarrhea. EaggEC are characterized by their adherence capacity that leads to aggregation of Hep-2 cells. This effect is associated with the presence of a virulence plasmid (pCVD432). EaggEC are supected to also produce a heat stable enterotoxin (EAST1) (49-53). They can belong to serogroups O44 and O126 (32).
Conventional detection methods for EHEC encompass enrichment and isolation with selective and/or indicator media such as E. coli broth, lauryl sulfate tryptose 4-methylumbelliferyl-b-acid broth, eosin methylene blue agar, McConkey sorbitol agar, and enterohemolysin agar (28,32,54-59). All of these assays, unfortunately, are indirect and lack the ability to identify EHEC or the other pathogenic E. coli strains specifically. Several methods for biochemical identification and immunological detection of EHEC have been put forward (54,60-63), however, it is well recognized that pathogenic E. coli strains neither posess nor lack unique fermentation pathways (58,64). Serotyping is not conclusive since no absolute correlation between serotype and pathogenic E. coli group can be established (12,27,32,58,65). DNA hybridization techniques have been established for experimental research but are not applicable for large scale routine diagnostic procedures (66,67). DNA amplification based assays, using PCR have been reported (68-72). Limitations to these methods include cumbersome post-PCR detection methods (agarose gel electrophoresis, Biotin/Avidin based ELISA detection systems).
To overcome these problems, a PCR assay which allows the specific determination of virulence factors characteristic for EHEC, ETEC, EPEC, EIEC, and EaggEC that is based on a fluorigenic detection method of PCR amplification has been developed.
This assay exploits the 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity of Taq-DNA polymerase (73) to cleave an internal oligonucleotide probe that is covalently conjugated with a fluorescent reporter dye (e.g. 6-carboxy-fluorescein [FAM]; xcexem=518 nm) and a fluorescent quencher dye (6-carboxytetram-ethyl-rhodamine [TAMRA]; xcexem=582 nm) at the 5xe2x80x2 and 3xe2x80x2 end, respectively (74,75). Fluorescence from FAM is efficiently quenched by TAMRA on the same, intact probe molecule (76). In the case that cognate PCR amplification occurs, Taq polymerase extends from the specific PCR primer and cleaves the internal, fluorigenic oligonucleotide probe annealed to the template strand. Thus, the reporter dye and the quencher dye get spatially separated. As a consequence of oligonucleotide hydrolysis and physical separation of the reporter and the quencher dyes, a measurable increase in fluoresecence intensity at 518 nm can be observed. PCR cycling leads to exponential amplification of the PCR product and consequently of fluorescence intensity.
TAQMAN(trademark)-PCR is performed in optical tubes that allow measurements of fluorescence signals without opening the PCR tubes. This dramatically minimizes post-PCR processing time and almost completely eliminates cross-PCR contamination problems. Employing this approach, simultaneous testing of biological materials for the presence of virulence genes of Lcoh strains and other enterobacteria, harboring virulence genes can be semiautomated and performed within 18 h.
According to the present invention Real Time PCR (e.g., TAQMAN(trademark) PCR) for the detection of pathogenic E. coli is provided, enabling for the first time the specific, rapid and high throughput routine detection of EHEC, ETEC, EPEC, EIEC, and EaggEC and related enterobacteria that harbor these virulence genes in routine bacteriological laboratories.
It is an object of the present invention to provide a rapid, high performance assay for the detection and identification of pathogenic E. coli in biological samples.
It is a further object of the present invention to provide specific, optimised primers and labelled oligonucleotide probes useful for the amplification of sequences encoding virulence factors/toxins characteristic for pathogenic E. coli 
The invention then, inter alia, comprises the following alone or in combination:
A method for the detection of pathogenic E. coli in a sample comprising PCR amplification of DNA isolated from said sample using a set of oligonucleotide primers specific for virulence factors/toxins of pathogenic E. coli selected from
primers that hybridise to a gene encoding heat labile toxin, or heat stabile toxin for the amplification of a DNA sequence characteristic for enterotoxigenic E. coli; 
primers that hybridise to a gene encoding heat stabile toxin for the amplification of a DNA sequence characteristic for enteroaggregative E. coli; 
primers that hybridise to the pCVD432 plasmid for the amplification of a DNA sequence characteristic for enteroaggregative E. coli; 
primers that hybridise to the inv-plasmid for the amplification of a DNA sequence contained in enteroinvasive E. coli; 
primers that hybridise to the EAF plasmid, or the eae gene for the amplification of a DNA sequence characteristic for enteropathogenic E. coli; and/ or
primers that hybridise to the genes encoding shiga-like toxin sltI or sltII for the amplification of a DNA sequence characteristic for enterohemorrhagic E. coli, followed by detection and identification of the amplified product using conventional methods;
the method as above wherein
the set of primers that hybridise to the gene encoding heat labile toxin characteristic for enterotoxigenic E. coli is
LT-1: 5xe2x80x2GCG TTA CTA TCC TCT CTA TGT G3xe2x80x2 (SEQ ID NO:1) and
LT-2: 5xe2x80x2AGT TTT CCA TAC TGA TTG CCG C3xe2x80x2 (SEQ ID NO:2);
the set of primers that hybridise to the gene encoding heat stabile toxin characteristic for enterotoxigenic E. coli is
ST-1: 5xe2x80x2TCC CTC AGG ATG CTA AAC CAG3xe2x80x2 (SEQ ID NO:3) and
ST-2a: 5xe2x80x2TCG ATT TAT TCA ACA AAG CAA C3xe2x80x2 (SEQ ID NO:4);
the set of primers that hybridise for the gene encoding heat stabile toxin characteristic for enteroaggregative E. coli is
EASTI-1: 5xe2x80x2AAC TGC TGG GTA TGT GGC TGG3xe2x80x2 (SEQ ID NO:5) and
EASTI-2: 5xe2x80x2TGC TGA CCT GCC TCT TCC ATC3xe2x80x2 (SEQ ID NO:6);
the set of primers which hybridise to the pCVD432 plasmid is
EA-1: 5xe2x80x2CTG GCG AAA GAC TGT ATC ATT G3xe2x80x2 (SEQ ID NO:7) and
EA-2: 5xe2x80x2TAA TGT ATA GAA ATC CGC TGT T3xe2x80x2 (SEQ ID NO:8);
the set of primers which hybridise to the inv-plasmid is
EI-1: 5xe2x80x2TTT CTG GAT GGT ATG GTG AGG3xe2x80x2 (SEQ ID NO:9) and
EI-2: 5xe2x80x2CTT GAA CAT AAG GAA ATA AAC3xe2x80x2 (SEQ ID NO:10);
the set of primers which hybridise to the EAF plasmid is
EP-1: 5xe2x80x2CAG GGT AAA AGA AAG ATG ATA AG3xe2x80x2 (SEQ ID NO:11) and
EP-2: 5xe2x80x2AAT ATG GGG ACC ATG TAT TAT C3xe2x80x2 (SEQ ID NO:12)
the set of primers which hybridise to the eae gene is
EPeh-1: 5xe2x80x2CCC GGA CCC GGC ACA AGC ATA AG3xe2x80x2 (SEQ ID NO:13) and
EPeh-2: 5xe2x80x2AGT CTC GCC AGT ATT CGC CAC C3xe2x80x2 (SEQ ID NO:14);
the primers which hybridises to the gene encoding shiga-like toxin SltI is
SltI-1: 5xe2x80x2ATG AAA AAA ACA TTA TTA ATA GC3xe2x80x2 (SEQ ID NO:15) and
SltI-2: 5xe2x80x2TCA CYG AGC TAT TCT GAG TCA AGC3xe2x80x2 (SEQ ID NO:16); and
the primers which hybridises to the gene encoding shiga-like toxin SltII is
SltII-1: 5xe2x80x2ATG AAG AAG ATR WTT RTD GCR GYT TTA TTY G3xe2x80x2 (SEQ ID NO:17) and
SltII-2: 5xe2x80x2TCA GTC ATW ATT AAA CTK CAC YTS RGC AAA KCC3xe2x80x2 (SEQ ID NO:18)
wherein W is A/T, R is A/G, D is A/G/T, Y is C/T and K is G/T; the method as above wherein a polymerase having additional 5xe2x80x2-3xe2x80x2 exonuclease activity is used for the amplification of DNA, and an oligonucleotide probe labelled at the most 5xe2x80x2 base with a fluorescent dye and at the most 3xe2x80x2 base with a fluorescent quencher dye which hybridises within the target DNA is included in the amplification process; said labelled oligonucleotide probe being susceptible to 5xe2x80x2-3xe2x80x2 exonuclease degradation by said polymerase to produce fragments that can be detected by fluorogenic detection methods;
the method as above wherein the labelled oligonucleotide probe for the detection of heat labile toxin characteristic for enterotoxigenic E. coli is
5xe2x80x2AGC TCC CCA GTC TAT TAC AGA ACT ATG3xe2x80x2 (SEQ ID NO:19);
the labelled oligonucleotide probe for the detection of heat stabile toxin characteristic for enterotoxigenic E. coli is
5xe2x80x2ACA TAC GTT ACA GAC ATA ATC AGA ATC AG3xe2x80x2 (SEQ ID NO:20);
the labelled oligonucleotide probe for the detection of heat stabile toxin characteristic for enteroaggregative E. coli is
5xe2x80x2ATG AAG GGG CGA AGT TCT GGC TCA ATG TGC3xe2x80x2 (SEQ ID NO:21);
the labelled oligonucleotide probe for the detection of pCVD432 plasmid is
5xe2x80x2CTC TTT TAA CTT ATG ATA TGT AAT GTC TGG3xe2x80x2 (SEQ ID NO:22);
the labelled oligonucleotide probe for the detection of the inv-plasmid is;
5xe2x80x2CAA AAA CAG AAG AAC CTA TGT CTA CCT3xe2x80x2 (SEQ ID NO:23)
the labelled oligonucleotide probe for the detection of the EAF-plasmid is;
5xe2x80x2CTT GGA GTG ATC GAA CGG GAT CCA AAT3xe2x80x2 (SEQ ID NO:24);
the labelled oligonucleotide probe for the detection of the eae gene is
5xe2x80x2TAA ACG GGT ATT ATC AAC AGA AAA ATC C3xe2x80x2 (SEQ ID NO:25);
the labelled oligonucleotide probe for the detection of shiga-like toxin SltI gene is
5xe2x80x2TCG CTG AAT CCC CCT CCA TTA TGA CAG GCA3xe2x80x2 (SEQ ID NO:26); and
the labelled oligonucleotide probe for the detection of shiga-like toxin SltII gene is
5xe2x80x2CAG GTA CTG GAT TTG ATT GTG ACA GTC ATT3xe2x80x2 (SEQ ID NO:27);
the method as above wherein the fluoroscent reporter dye is 6-carboxy-fluoroscein, tetrachloro-6-carboxy-fluoroscein, or hexachloro-6-carboxy-fluoroscein, and the fluorescent quencher dye is 6-carboxytetramethyl-rhodamine;
the method as above wherein the PCR amplification process consists of 35 PCR cycles at a MgCl2 concentration of 5.2 mmol, an annealing temperature of 55xc2x0 C. and an extension temperature of 65xc2x0 C.;
a set of primers useful for PCR amplification of DNA specific for virulence factors/toxins of pathogenic E. coli selected from:
a set of primers that hybridise to a gene encoding heat labile toxin, or heat stabile toxin of enterotoxigenic E. coli; 
a set of primers that hybridise to a gene encoding heat stabile toxin of enteroaggregative E. coli; 
a set of primers that hybridise to the pCVD432 plasmid of enteroaggregative E. coli; 
a set of primers that hybridise to the inv-plasmid of enteroinvasive E. coli; 
a set of primers that hybridise to the EAF plasmid, or the eae gene of enteropathogenic E. coli; and
a set of primers that hybridise to the gene encoding shiga-like toxin sltI or sltII of enterohemorrhagic E. coli; 
the set of primers as above wherein
the set of primers which hybridise to the gene encoding heat labile toxin of enterotoxigenic E. coli is
LT-1: 5xe2x80x2GCG TTA CTA TCC TCT CTA TGT G3xe2x80x2 (SEQ ID NO:1) and LT-2: 5xe2x80x2AGT TTT CCA TAC TGA TTG CCG C3xe2x80x2 (SEQ ID NO:2);
the set of primers which hybridise to the gene encoding heat stabile toxin of enterotoxigenic E. coli is
ST-1: 5xe2x80x2TCC CTC AGG ATG CTA AAC CAG3xe2x80x2 (SEQ ID NO:3) and
ST-2a: 5xe2x80x2TCG ATT TAT TCA ACA AAG CAA C3xe2x80x2 (SEQ ID NO:4);
the set of primers which hybridise to the gene encoding heat stabile toxin of enteroaggregative E. coli is
EASTI-1: 5xe2x80x2AAC TGC TGG GTA TGT GGC TGG3xe2x80x2 (SEQ ID NO:5) and
EASTI-2: 5xe2x80x2TGC TGA CCT GCC TCT TCC ATG3xe2x80x2 (SEQ ID NO:6);
the set of primers which hybridise to the pCVD432 plasmid is
EA-1: 5xe2x80x2CTG GCG AAA GAC TGT ATC ATT G3xe2x80x2 (SEQ ID NO:7) and
EA-2: 5xe2x80x2TAA TGT ATA GAA ATC CGC TGT T3xe2x80x2 (SEQ ID NO:8);
the set of primers which hybridise to the inv-plasmid is
EI-1: 5xe2x80x2TTT CTG GAT GGT ATG GTG AGG3xe2x80x2 (SEQ ID NO:9) and
EI-2: 5xe2x80x2CTT GAA CAT AAG GAA ATA AAC3xe2x80x2 (SEQ ID NO:10);
the set of primers which hybridise to the EAF plasmid is
EP-1: 5xe2x80x2CAG GGT AAA AGA AAG ATG ATA AG3xe2x80x2 (SEQ ID NO:11) and
EP-2: 5xe2x80x2AAT ATG GGG ACC ATG TAT TAT C3xe2x80x2 (SEQ ID NO:12);
the set of primers which hybridise to the eae gene is
EPeh-1: 5xe2x80x2CCC GGA CCC GGC ACA AGC ATA AG3xe2x80x2 (SEQ ID NO:13) and
EPeh-2: 5xe2x80x2AGT CTC GCC AGT ATT CGC CAC C3xe2x80x2 (SEQ ID NO:14);
the set of primers which hybridise to the shiga-like toxin sltI gene is
SltI-1: 5xe2x80x2ATG AAA AAA ACA TTA TTA ATA GC3xe2x80x2 (SEQ ID NO:15) and
SltI-2: 5xe2x80x2TCA CYG AGC TAT TCT GAG TCA AGC3xe2x80x2 (SEQ ID NO:16);
and
the set of primers which hybridise to the shiga-like toxin sltII is
SltI-1: 5xe2x80x2ATG AAG AAG ATR WTT RTD GCR GYT TTA TTY G3xe2x80x2 (SEQ ID NO:17) and
SltII-2: 5xe2x80x2TCA GTC ATW ATT AAA CTK CAC YTS RGC AAA KCC3xe2x80x2 (SEQ ID NO:18)
wherein W is A/T, R is A/G, D is A/G/T, Y is C/T and K is G/T;
the set of primers as above which in addition to the primers for amplification of target DNA comprise a labelled oligonucleotide probe which is labelled with a fluoroscent reporter dye, such as 6-carboxy-fluoroscein, tetrachloro-6-carboxy-fluoroscein, hexachloro-6-carboxy-fluoroscein, at the most 5xe2x80x2 base and a fluoroscent quencher dye, such as 6-carboxytetramethyl-rhodamine, at the most 3xe2x80x2 base, and have a nucleotide sequence selected from
5xe2x80x2AGC TCC CCA GTC TAT TAC AGA ACT ATG3xe2x80x2 (SEQ ID NO:19)
which hybridises to a gene encoding heat labile toxin of enterotoxigenic E. coli;
5xe2x80x2ACA TAC GTT ACA GAC ATA ATC AGA ATC AG3xe2x80x2 (SEQ ID NO:20)
which hybridises to a gene encoding heat stabile toxin of enterotoxigenic E. coli;
5xe2x80x2ATG AAG GGG CGA AGT TCT GGC TCA ATG TGC3xe2x80x2 (SEQ ID NO:21)
which hybridises to a gene encoding heat stabile toxin of enteroaggregative E. coli;
5xe2x80x2CTC TTT TAA CTT ATG ATA TGT AAT GTC TGG3xe2x80x2 (SEQ ID NO:22)
which hybridises to the pCVD432 plasmid;
5xe2x80x2CAA AAA CAG AAG AAC CTA TGT CTA CCT3xe2x80x2 (SEQ ID NO:23)
which hybridises to the inv-plasmid;
5xe2x80x2CTT GGA GTG ATC GAA CGG GAT CCA AAT3xe2x80x2 (SEQ ID NO:24)
which hybridises to the EAF plasmid;
5xe2x80x2TAA ACG GGT ATT ATC AAC AGA AAA ATC C3xe2x80x2 (SEQ ID NO:25)
which hybridises to the eae gene;
5xe2x80x2TCG CTG AAT CCC CCT CCA TTA TGA CAG GCA3xe2x80x2 (SEQ ID NO:26)
which hybridises to the shiga-like toxin SltI gene; and
5xe2x80x2CAG GTA CTG GAT TTG ATT GTG ACA GTC ATT3xe2x80x2 (SEQ ID NO:27)
which hybridises to the shiga-like toxin SltII gene;
the use of the method as above for diagnosing an E. coli infection of a living animal body, including a human, or for the detection of E. coli contamination of consumables, such as meat, milk and vegetables.
Conventional methods used to detect PCR amplification are laboursome, employ potentially carcinogenic substances (ethidium bromide gel electrophoresis), and are not suited as a routine assay method in the microbiological routine laboratory (68-72). This poses a serious problem, especially when potential pathogenic bacteria cannot be differentiated from facultative pathogenic or apathogenic ones due to characteristic biochemical, serological and/or morphological criteria. Thus, specific nucleic acid-based diagnostic methods that directly detect virulence factors or toxins harbored by these species are mandatory. This is in principal the case for the diagnosis of pathogenic E. coli bacteria. Biochemical properties of EHEC, EPEC, EIEC, ETEC, and EaggEC are not unique and cannot be used for setting them apart from other E. coli strains (54,60-62). Furthermore, virulence plasmids of E. coli can be found in other enterobacteria as well (38,48,83,88,89). Because of the diverse serological makeup, identification of pathogenic E. coli by serotyping is also not an accurate means of identification (12,15,28-32). Classical colony hybridization assays with probes specific for characteristic virulence factor and/or toxin genes are laborous and timeconsuming (66,67). Classical PCR methods require various post-PCR steps in order to verify whether specific amplification of a target gene has occured (68J2). The TAQMAN(trademark)-PCR detection system (74,75,90) enables the rapid, specific, sensitive, and high-throughput diagnosis for differentiation of pathogenic Lcoli strains from other strains of E. coli. The assay has the ability to quantify the initial target sequence. Since PCR-reaction tubes have not to be opened after PCR cycling, the potential danger of cross-PCR contamination is almost negligible. The scanning time of 96 samples is approximately 8 min, and calculation of test results can be automated with a commercially available spread sheet program. Thus, overall post-PCR processing time is cut to a minimum.
The TAQMAN(trademark)-System relies on standard PCR technique with the addition of a specific internal fluorogenic oligonucleotide probe. The combination of conventional PCR with the Taq polymerase-dependent degradation of an internally hybridized ohgonucleotide probe confers also specificity to this detection method, since it is highly unlikely that unspecific PCR amplification will yield positive fluorescence signals. Some rules for chosing the fluorigenic probes have to be obeyed (74,75). Criticial are the lenght of the probe, the location of reporter and quencher dyes and the absence of a guanosine at the 5xe2x80x2-end (74). Also, the distance of the probe from one of the specific PCR primers is important. This is due to the fact that the probe has to stay annealed to the template strand in order to be cleaved by Taq polymerase. Since annealing depends, at least partially, on the Tm of the probe, probes should be designed to have a higher Tm as the primers. According to the present invention this was solved (except for sltII) by designing probes that were 3 to 6 bp longer than the specific primers. PCR amplification includes extension of the target sequence after annealing of the primers and the Tm of the extended primers increases. For the fluorogenic oligonucleotide probe, where the 3xe2x80x2- end is capped in order to avoid elongation, the Tm remains constant, making it more likely that the probe dissociates before degradation by Taq polymerase. Oligonucleotide probe degradation can be optimized by spatial proximity of the fluorogenic probe and the primer. By moving the probe for SltI from 121 bp to 9 bp close to the primer, a significant improvement in ARQ values could be obtained. A second strategy of optimization of TAQMAN(trademark)-PCR is to perform PCR elongation at 65xc2x0 C., where it is also less likely that the probe dissociates from the template strand before Taq polymerase reaches and hydrolizes it. Values for xcex94RQ can thus again be increased about 1.2 to 1.5 fold. The increase of xcex94RQ values might be due to the ratio of annealed oligonucleotide probe reached by Taq polymerase or to an increased processivity of Taq polymerase.
The concentration of fluorogenic probes influences the accuracy of TAQMAN(trademark)-results. When the probe concentrations were  greater than 50 pmol/PCR reaction only a relatively small fraction was hydrolysed by Taq polymerase. The ratio of undegraded probe to degraded probe remains high and the fluorescence emmission of the unquenched reporter dye does not significantly increase in relation to the fluorescence intensity of the reporter dye sill close to the quencher. Thus, at high probe concentrations, ARQ values are lower than with intermediate probe concentrations (10 20 pmol). When the probe concentration is too low, xcex94RQ values are increased, however, variability of PCR results is increased, since probably small errors in pipeting or minimal differences between PCR reactions become critical. Optimal probe concentration that yielded smallest variabilties and highest RQ values were found at a probe concentration of 20 pmol.
Since TAQMAN(trademark)-PCR uses an internal oligonucleotide probe for detection of template amplification, specific primers and probes can be amply designed. The design of primer and probe sequences is especially important, when nucleotide sequence variants of a given gene exist. This is the case for sltI and sltII. For sltI, all published sequences were aligned and primers and probes were designed to bind to conserved regions of all three variants. For sltII, only one region of the published genes was conserved, thus this region was chosen for the fluorogenic oligonucleotide probe. The primers for amplification of sltII were designed to contain all possible nucleotide sequences at the ambiguous positions of the published sltll variants (degenerate primer approach) (79-83). By employing degenerate primers, it is possible to detect all published variants in one single PCR reaction.
The isolation method for template DNA affects the performance of the PCR. Two methods, that are suited as rapid purification steps for routine applications, namely boiling prep or spin prep were compared. Boiling preps may still contain some bacterial components that can affect PCR reactions, however, it is extremely fast. The spin prep method involves isolation steps that serve to purify DNA from potentially negatively influencing materials. xcex94RQ values and sensitivity of TAQMAN(trademark)-PCR for virulence genes from enterobacteria was not found significantly increased as compared to boiling preps when template DNA was prepared by spin prep method. The overall sensitivity of TAQMAN(trademark)-PCR for all primer/probe combinations was comparable to visual scoring of PCR products by detection with ethidium bromide stained agarose gel electrophoresis. Under optimized. conditions, as few as 103 cfu sltI+EHEC could be detected among 107 non-pathogenic E. coli per PCR reaction.
The use of immunomagnetic detection methods for E. coli O157 (54,91) has been put forward as a means to improve sensitivity of EHEC diagnostics by enrichment of this serogroup since the first sit producing strains were found to be O157:H7 positive (1,2). However, it is obvious that EHEC that are O157 antigen negative will be missed by this method. It became clear during serotyping studies of recent EHEC isolates that the number of O157+EHEC now is small as compared to non-O157 EHEC (12,15,28,29,31). In a recent study, conducted in Southern Germany only 2 of 13 isolates were O157 positive (92). Immunomagnetic detection methods for other O serotypes are currently not available. Also, other enterobacteria such as Citrobacter sp. (83) and Enterobacter sp. (89) that can harbor shiga like toxins would be missed in the case of biased enrichment procedures previous to analysis of virulence genes. Thus, TaqMan(trademark)-based PCR that is designed for detection of virulence genes in all enterobacteria appears to be superior.
The infectious agents of a large proportion of diarrheal diseases is not known. Routine screening for bacterial pathogens in the gastrointestinal tract encompasses Salmonella sp, Shigella sp, S. aureus, Campylobacter sp., Vibrio sp., Yersinia sp., and C. difficile (32). It is well recognized that pathogenic E. coli such as ETEC, EHEC, EIEC, and EaggEC are important pathogens of the lower gastrointestinal tract and therefore might significantly contribute to the number of diarrheal infections (32). However, no routine bacteriological diagnostic procedures for these bacteria are performed, and, moreover, in most cases these pathogenic E. coli are misdiagnosed under the category of non-pathogenic xe2x80x9ccommensal floraxe2x80x9d. In order to address this problem a set of specific primers and fluorogenic probes were developed and optimized for TAQMAN(trademark)-based detection of virulence factors harbored by these bacteria (Tables 2 and 3). Arranging patient samples, positive and no-template controls of all 8 tested virulence genes in a standard 96 well microtiter format, a turnaround time from preparation of sample DNA to fluorescence measurement of under 5 hours can be achieved. Thus, the TAQMAN(trademark)-based assay for pathogenic E. coli provides an ultrarapid means of diagnosis of these bacteria. While being accurate, sensitive and specific, this assay requires minimal post PCR processing time compared to conventional methods. When TAQMAN(trademark)-PCR is performed in optical tubes also the danger of cross-contamination of PCR reactions with amplified products is reduced to a minimum. Detection of virulence plasmids harbored by pathogenic enterobacteria might prove the potential of these bacteria to cause disease in the host. It is not clear whether enterobacteria that contain toxin genes or attachment factors do also always express them outside the host. This might be an explanation why ELISA tests for shiga like toxins might be negative in a number of HUS cases where sltI and/or sltII containing EHECs can be detected by nucleic acid based methods.
The TAQMAN(trademark)-assay according to the invention for detection of pathogenic E. coli was then tested in a routine diagnostic setting for the examination of stool samples obtained from children with diarrhea within a defined geographic area (Southern Bavaria) during a 7 month period. Results obtained by TAQMAN(trademark)-PCR were compared to the standard detection method for PCR products (electrophoresis of ethidium stained agarose gels). 100 stool samples were analysed (Table 4). 22% of samples were found to test positive for one or more virulence factors. There were 2 cases. of EHEC, 5 ETEC, 8 EaggEC, 1 EIEC, and 16 EPEC. This means that ⅕ of children with diarrhea probably suffered from diarrhea caused by pathogenic E. coli. These numbers are far higher than these for all other groups of routinely screened bacterial gastrointestinal tract pathogens. Only 2 cases of salmonella and no campylobacter were observed within this group.
Interestingly, the two children diagnosed with EHEC were severely sick, one suffered from hemorrhagic colitis, the other developed HUS and had to be treated in a critical care unit.
Collectively, these investigations show that a large proportion of diarrheal diseases in children and also in adults are associated with pathogenic E. coli that are falsely diagnosed as commensal flora in standard microbiological procedures. The TAQMAN(trademark) methodology according to the invention for the first time enables the direct, fast, specific, and sensitive detection of these important pathogens. Moreover, virulence genes detected with this approach are not confined to E. coli, they also can be freely transmitted to other enterobacteria. Detection of the virulence genes within these bacteria would also be covered by the herein described TAQMAN(trademark)-PCR The assay requires only minimal post-PCR detection time, can thus be performed under 18 hours, and abolishes PCR-cross contamination problems.
According to the present invention E. coli virulence factor/toxin genes were used as targets for PCR amplification. PCR primers and fluorogenic probes were designed on the basis of published sequences. Eight different primer and probe sets for detection of pathogenic groups of E. coli and related enterobacteria were specifically chosen, see table 1.
Primer sequences and their locations with GenBank accessions are detailed in Table 2. Detection of EHEC sltI is based on consensus primer and probe sequences after alignment of sltI homologous genes (Genbank accessions Z36899, Z36900, and Z36901) (77,78). Detection of sltII variants is based on published sequences of homologous genes (Genbank accessions M76738, Z37725, L11079, X67515, M59432, M29153, M36727, and M21534) (79-83). For amplification of sltII, degenerate primer sets proved optimal. Diagnosis of ETEC is based on amplification of either heat labile (LT) (84) or heat stable toxin (ST) (36), EaggEC on pCVD432 plasmid sequences (40,50), EIEC on inv-plasmid sequences (38,48), EPEC on E. coli attaching and effacing gene (EAF plasmid) (37,85) or E. coli gene for EHEC attaching and effacing protein (eae) (86). PCR control amplification for integrity of DNA preparations was performed using primers specific for the E. coli parC gene (topoisomerase IV, Genbank accession M58408) (87).
Oligonucleotide probes and their Genbank Ref. are shown in table 3. Oligonucleotide probes were designed (if possible) with a GC-content of 40-60%, no G-nucleotide at the 5xe2x80x2-end, length of probes was 27 to 30 bp. Probes were covalently conjugated with a fluorescent reporter dye (e.g. 6-carboxy-fluorescein [FAM]; xcexem=518 nm) and a fluorescent quencher dye (6-carboxytetram-ethyl-rhodamine [TAMRA]; xcexem=582 nm) at the most 5xe2x80x2 and most 3xe2x80x2 base, respectively. All primers and probes were obtained from Perkin Elmer, Germany.
TAQMAN(trademark)-PCR was optimized by isolation of DNA from E. coli control strains harboring genes for LT, ST, inv-plasmid, pCVD342, EAF, eae, sltI and sltII (see Table 1). MgCl2 concentrations were adjusted for maximum PCR. product yields (as verified by agarose gel electrophoresis) and RQ values (RQ=FAMfluorescence intensity/TAMRAfluorescence intensity) with the above mentioned pathogenic E. coli control strains. Optimum PCR reactions for all primer/fluorigenic probes used were obtained at a MgCl2 concentration of 5.2 mmol, 35 PCR cycles, an annealing temperature of 55xc2x0 C. and an extension temperature of 65xc2x0 C. Extension at 65xc2x0 C. was found to yield higher RQ values, probably due to a lower rate of template/fluorogenic probe dissociation before degradation by Taq-polymerase.
The E. coli sltI gene was used as a target sequence for establishment of PCR and analysing different locations of probes relative to the PCR primers. Primers were designed to anneal in conserved regions of the sltI genes (see above). Two probes, sltI-N0 located 132 bp upstream of one primer and sltI-N1, placed at a 21 bp distance from the primer were compared. RQ values achieved with probe sltI-N1(RQm=6.3800) were reproducably found higher than RQ values generated with probe sltI-NO (RQm=0.9620) at equal template concentrations of the E. coli sltI control DNA. Generally, also probes specific for other target genes that were located close (4 to 20 bp) to one of the two PCR primers yielded consistently higher RQ values than probes that were placed at a greater distance from the primers.
The influence of DNA preparation on the performance of TAQMAN(trademark)-PCR was tested, since it has been reported that crude bacterial lysates can contain inhibiting factors that might interfere with PCR performance. Therefore, bacteria were collected after overnight growth on McConkey plates. DNA was prepared by boiling of bacteria inoculated in 0.9% NaCl solution or by isolation of genomic DNA with a commercial spin prep procedure (see the example, material and methods). The RQ values and sensititvity of TAQMAN(trademark)-PCR did not differ when the two preparation methods were compared. The RQ values obtained for PCR amplifications from DNA derived from 105 Sltl or sltII containing EHEC prepared by boiling or by spin prep comparable.
The TAQMAN(trademark)-PCR method relies on the detection of free reporter dye (FAM) that is released from the probe after hydrolysis. Thus, probe concentration should also have an effect on the assay performance by affecting the fraction of the probe that is degraded during PCR cycling. Probe concentrations were titrated in the range of 100 pmol to 0.1 pmol and xcex94RQ values were determined. Optimal probe concentrations varied in between 10 pmol and 20 pmol depending on the target gene that was amplified.
For testing sensitivity of TAQMAN(trademark)-PCR, EHEC containing either sltl or sltll were diluted in a suspension containing E. coli strain ATCC11775 at 107 CfU at log step dilutions. PCR was performed under optimized conditions and results from ethidium-bromide stained agarose gels were compared to TaqMan(trademark) results. Minimum detection limits of a sltl containing EHEC strain was 103 cfu within 107. For sltII the detection limit was found at 103.5 cfu in 107 enterobacteria. Both methods, detection of PCR products by agarose gel electrophoresis and measurement of fluorescence signals by the TaqMan method yielded comparable results, i.e. that at xcex94RQ values above xcex94RQthreshold PCR product bands were visible in agarose gelb whereas at xcex94RQ values around xcex94RQ threshold also in agarose gels PCR products were below the detection limit. After optimizing detection tests for all virulence factors/toxins, TAQMAN(trademark)-PCR was set up for routine testing of biological specimen for the presence of pathogenic E. coli bacteria. Results of TAQMAN(trademark)-PCR were compared to agarose gel electrophoresis.
The following example will illustrate the invention further. It is, however, not to be construed as limiting.